The use of human-induced pluripotent cell lines in the differentiation of specific cell types and assay development has in recent years progressed significantly. In this introduction to the field, the rationale for using induced pluripotent stem cells in drug development is described and some of the advantages and drawbacks are discussed. The prospect of facilitating drug development by using stratified sources of cells from diverse patient groups and differentiating these to organ and tissue cells for their use in disease models, screening assays, and ADME testing is highlighted. The variety of methodologies and protocols presented in this volume provides a selected collection of experiences and practice. Here, these are discussed in a general perspective with references and comments to other similar protocols in the literature.

The ideal cell culture model should mimic the cell’s physiology and the mechanical and chemical cues that are present in specific tissues and organs, within a convenient high-throughput format. A possible key feature for such models is to recapture the cell polarity, the interactions between cells, and the interactions between the cells and the elastic extracellular matrix (ECM) by orienting the cells in a three-dimensional (3D) matrix. A common method to create 3D cell environments is to let the cells aggregate into spheroids with a diameter of around 200 μm. A major challenge for 3D cell cultures is to perform quick and easy imaging of the dense cell population, especially non-invasively. This protocol explains how to take advantage of the number of cells growing out from cell spheroids over time as a readout of the effect of a drug. The assay is carried out in a microphysiological system where the cells experience in vivo-like fluidic conditions. The procedure is compatible with standard imaging techniques and can be performed non-invasively using light microscopy or as a complement to other fluorescent imaging assays.

Microphysiological systems (MPS), i.e., miniaturized mimics of active biological systems, are considered resourceful tools for facilitating drug development. It presupposes that a microphysiological system can respond as organs and tissues in the human body to new drug candidates and that relevant cellular response can be monitored. If, or when, this is possible, microphysiological systems have the potential to support and accelerate the development and testing of new drug candidates. So far, the focus has been more on the design and fabrication of the microarchitecture of the MPS to recreate their cellular counterparts as closely and realistically as possible. Commercial MPS devices have been developed that focus on advanced technological design solutions, sometimes more than their actual biological functionality. This chapter discusses in brief terms the prerequisites for the design and construction of MPS devices in relation to their utility for drug development and testing in a more holistic manner. The use of induced pluripotent cells as a prime source for mimicking the in vivo conditions of diseased conditions is mainly considered. Existing limitations and needs are highlighted in relation to the space for improvements, based on recently published results.

Polyploid giant cancer cells (PGCCs) contribute to the genetic heterogeneity and evolutionary dynamics of tumors. Their size, however, complicates their isolation from mainstream tumor cell populations. Standard techniques like fluorescence-activated cell sorting (FACS) rely on fluorescent labeling, introducing potential challenges in subsequent PGCC analyses. In response, we developed the Isosceles Trapezoidal Spiral Microchannel (ITS mu C), a microfluidic device optimizing the Dean drag force (FD) and exploiting uniform vortices for enhanced separation. Numerical simulations highlighted ITS mu C's advantage in producing robust FD compared to rectangular and standard trapezoidal channels. Empirical results confirmed its ability to segregate larger polystyrene (PS) particles (avg. diameter: 50 mu m) toward the inner wall, while directing smaller ones (avg. diameter: 23 mu m) outward. Utilizing ITS mu C, we efficiently isolated PGCCs from doxorubicin-resistant triple-negative breast cancer (DOXR-TNBC) and patient-derived cancer (PDC) cells, achieving outstanding purity, yield, and viability rates (all greater than 90%). This precision was accomplished without fluorescent markers, and the versatility of ITS mu C suggests its potential in differentiating a wide range of heterogeneous cell populations. Polyploid giant cancer cells (PGCCs) contribute to the genetic heterogeneity and evolutionary dynamics of tumors.

Production of therapeutic monoclonal antibodies (mAbs) is a complex process that requires extensive analytical and bioanalytical characterization to ensure high and consistent product quality. Aggregation of mAbs is common and very problematic and can result in products with altered pharmacodynamics and pharmacokinetics and potentially increased immunogenicity. Rapid detection of aggregates, however, remains very challenging using existing analytical techniques. Here, we show a real-time and label-free fiber optical nanoplasmonic biosensor system for specific detection and quantification of immunoglobulin G (IgG) aggregates exploiting Protein A mediated avidity effects. Compared to monomers, IgG aggregates were found to have substantially higher apparent affinity when binding to Protein Afunctionalized sensor chips in a specific pH range (pH 3.8-4.0). Under these conditions, aggregates and monomers showed significantly different binding and dissociation kinetics. Reliable and rapid aggregate quantification was demonstrated with a limit of detection (LOD) and limit of quantification (LOQ) of about 9 and 30 mu g/mL, respectively. Using neural network-based curve fitting, it was further possible to simultaneously quantify monomers and aggregates for aggregate concentrations lower than 30 mu g/mL. Our work demonstrates a unique avidity-based biosensor approach for fast aggregate analysis that can be used for rapid at-line quality control, including lot/batch release testing. This technology can also likely be further optimized for real-time in-line monitoring of product titers and quality, facilitating process intensification and automation.

Therapeutic monoclonal antibodies (mAbs) provide new means for treatments of a wide range of diseases and comprise a large fraction of all new approved drugs. Production of mAbs is expensive compared to conventional drug production, primarily due to the complex processes involved. The affinity purification step is dominating the cost of goods in mAb manufacturing. Process intensification and automation could reduce costs, but the lack of real-time process analytical technologies (PAT) complicates this development. We show a specific and robust fiber optical localized surface plasmon resonance (LSPR) sensor technology that is optimized for in-line product detection in the effluent in affinity capture steps. The sensor system comprises a flow cell and a replaceable sensor chip functionalized with biorecognition elements for specific analyte detection. The high selectivity of the sensor enable detection of mAbs in complex sample matrices at concentrations below 2.5 mu g mL(-1). In place regeneration of the sensor chips allowed for continuous monitoring of multiple consecutive chromatographic separation cycles. Excellent performance was obtained at different purification scales with flow rates up to 200 mL min(-1). This sensor technology facilitates efficient column loading, optimization, and control of chromatography systems, which can pave the way for continuous operation and automation of protein purification steps.

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In situ capacitance sensing is shown to be capable of monitoring critical morphological changes in industrial cultures by analyzing the sensor’s frequency spectrum. Scanning the frequency of an alternating current between the electrodes of a capacitance sensor, placed in a cell culture, allowed detection of the size change of microbial cells from shifts in the spectra. The frequency was scanned between 0.1–15 MHz and cell size was measured from 1 to 20 μm. The analysis of the spectra was verified with two recombinant strains, one producing human insulin and another Green Fluorescence Protein (GFP). Both the insulin and GFP cultivations were carried out in 6 L fed-batch bioreactors using typical industrial procedures. The spectral analysis provided critical information about the changes in the size of the cells. It is suggested that this information may have high relevance for a better assessment of the state of cultivations producing proteins, for optimization and for improving the economy of large-scale biopharmaceutical production.

There is a profound need in bioprocess manufacturing for low-cost single-use sensors that allow timely monitoring of critical product and production attributes. One such opportunity is screen-printed enzyme-based electrochemical sensors, which have the potential to enable low-cost online and/or off-line monitoring of specific parameters in bioprocesses. In this study, such a singleuse electrochemical biosensor for lactate monitoring is designed and evaluated. Several aspects of its fabrication and use are addressed, including enzyme immobilization, stability, shelf-life and reproducibility. Applicability of the biosensor to off-line monitoring of bioprocesses was shown by testing in two common industrial bioprocesses in which lactate is a critical quality attribute (Corynebacterium fermentation and mammalian Chinese hamster ovary (CHO) cell cultivation). The specific response to lactate of the screen-printed biosensor was characterized by amperometric measurements. The usability of the sensor at typical industrial culture conditions was favorably evaluated and benchmarked with commonly used standard methods (HPLC and enzymatic kits). The single-use biosensor allowed fast and accurate detection of lactate in prediluted culture media used in industrial practice. The design and fabrication of the biosensor could most likely be adapted to several other critical bioprocess analytes using other specific enzymes. This makes this single-use screen-printed biosensor concept a potentially interesting and versatile tool for further applications in bioprocess monitoring. © 2020 by the authors.